Defining the 3'Epigenetic Boundary of the FMR1 Promoter and Its Loss in Individuals with Fragile X Syndrome.

This study characterizes the DNA methylation patterns specific to fragile X syndrome (FXS) with a full mutation (FM > 200 CGGs), premutation (PM 55-199 CGGs), and X inactivation in blood and brain tissues at the 3' boundary of the FMR1 promoter. Blood was analyzed from 95 controls and 462 individuals (32% males) with FM and PM alleles. Brain tissues (62% males) were analyzed from 12 controls and 4 with FXS. There was a significant increase in intron 1 methylation, extending to a newly defined 3' epigenetic boundary in the FM compared with that in the control and PM groups (p < 0.0001), and this was consistent between the blood and brain tissues. A distinct intron 2 site showed a significant decrease in methylation for the FXS groups compared with the controls in both sexes (p < 0.01). In all female groups, most intron 1 (but not intron 2 sites) were sensitive to X inactivation. In all PM groups, methylation at the 3' epigenetic boundary and the proximal sites was significantly decreased compared with that in the control and FM groups (p < 0.0001). In conclusion, abnormal FMR1 intron 1 and 2 methylation that was sensitive to X inactivation in the blood and brain tissues provided a novel avenue for the detection of PM and FM alleles through DNA methylation analysis.

Relationships between age and epi-genotype of the FMR1 exon 1/intron 1 boundary are consistent with non-random X-chromosome inactivation in FM individuals, with the selection for the unmethylated state being most significant between birth and puberty.

Methylation of the fragile X-related epigenetic element 2 (FREE2) located on the exon 1/intron 1 boundary of the FMR1 gene is related to FMRP expression and cognitive impairment in full mutation (FM; CGG>200) individuals. We examined the relationship between age, the size of the FMR1 CGG expansion and the methylation output ratio (MOR) at 12 CpG sites proximal to the exon 1/intron 1 boundary using FREE2 MALDI-TOF MS. The patient cohort included 119 males and 368 females, i.e. 121 healthy controls (CGG<40), 176 premutation (CGG 55-170) and 190 FM (CGG 213-2000). For all CpG units examined, FM males showed a significantly elevated MOR compared with that in hypermethylated FM females. In FM males the MOR for most CpG units significantly positively correlated with both age and CGG size (P< 0.05). In FM females the skewing towards the unmethylated state was significant for half of the units between birth and puberty (P < 0.05). The methylation status of intron 1 CpG10-12 that was most significantly related to cognitive impairment in our earlier study, did not change significantly with age in FM females. These results challenge the concept of fragile X syndrome (FXS)-related methylation being static over time, and suggest that due to the preference for the unmethylated state in FM females, X-inactivation at this locus is not random. The findings also highlight that the prognostic value of FXS methylation testing is not uniform between all CpG sites, and thus may need to be evaluated on a site-by-site basis.

Detection of skewed X-chromosome inactivation in Fragile X syndrome and X chromosome aneuploidy using quantitative melt analysis.

Methylation of the fragile X mental retardation 1 (FMR1) exon 1/intron 1 boundary positioned fragile X related epigenetic element 2 (FREE2), reveals skewed X-chromosome inactivation (XCI) in fragile X syndrome full mutation (FM: CGG > 200) females. XCI skewing has been also linked to abnormal X-linked gene expression with the broader clinical impact for sex chromosome aneuploidies (SCAs). In this study, 10 FREE2 CpG sites were targeted using methylation specific quantitative melt analysis (MS-QMA), including 3 sites that could not be analysed with previously used EpiTYPER system. The method was applied for detection of skewed XCI in FM females and in different types of SCA. We tested venous blood and saliva DNA collected from 107 controls (CGG < 40), and 148 FM and 90 SCA individuals. MS-QMA identified: (i) most SCAs if combined with a Y chromosome test; (ii) locus-specific XCI skewing towards the hypomethylated state in FM females; and (iii) skewed XCI towards the hypermethylated state in SCA with 3 or more X chromosomes, and in 5% of the 47,XXY individuals. MS-QMA output also showed significant correlation with the EpiTYPER reference method in FM males and females (P < 0.0001) and SCAs (P < 0.05). In conclusion, we demonstrate use of MS-QMA to quantify skewed XCI in two applications with diagnostic utility.

Normal DNA methylation dynamics in DICER1-deficient mouse embryonic stem cells.

Reduced DNA methylation has been reported in DICER1-deficient mouse ES cells. Reductions seen at pericentric satellite repeats have suggested that siRNAs are required for the proper assembly of heterochromatin. More recent studies have postulated that the reduced methylation is an indirect effect: the loss of Mir290 cluster miRNAs leads to upregulation of the transcriptional repressor RBL2 that targets the downregulation of DNA methyltransferase (Dnmt) genes. However, the observations have been inconsistent. We surmised that the inconsistency could be related to cell line "age," given that DNA methylation is lost progressively with passage in DNMT-deficient ES cells. We therefore subjected Dicer1(-/-) ES cells to two experimental regimes to rigorously test the level of functional DNMT activity. First, we cultured them for a prolonged period. If DNMT activity was reduced, further losses of methylation would occur. Second, we measured their DNMT activity in a rebound DNA methylation assay: DNA methylation was stripped from Cre/loxP conditionally mutant Dicer1 ES cells using a shRNA targeting Dnmt1 mRNA. Cre expression then converted these cells to Dicer1(-/-), allowing for DNMT1 recovery and forcing the cells to remethylate in the absence of RNAi. In both cases, we found functional DNMT activity to be normal. Finally, we also show that the level of RBL2 protein is not at excess levels in Dicer1(-/-) ES cells as has been assumed. These studies reveal that reduced functional DNMT activity is not a salient feature of DICER1-deficient ES cells. We suggest that the reduced DNA methylation sometimes observed in these cells could be due to stochastic alterations in DNA methylation patterns that could offer growth or survival advantages in culture, or to the dysregulation of pathways acting in opposition to the DNMT pathway.

Early detection of fragile X syndrome: applications of a novel approach for improved quantitative methylation analysis in venous blood and newborn blood spots.

BACKGROUND: Standard fragile X syndrome (FXS) diagnostic tests that target methylation of the fragile X mental retardation 1 (FMR1) CpG island 5' of the CGG expansion can be used to predict severity of the disease in males from birth, but not in females. METHODS: We describe methylation specific-quantitative melt analysis (MS-QMA) that targets 10 CpG sites, with 9 within FMR1 intron 1, to screen for FXS from birth in both sexes. The novel method combines the qualitative strengths of high-resolution melt and the high-throughput, quantitative real-time PCR standard curve to provide accurate quantification of DNA methylation in a single assay. Its performance was assessed in 312 control (CGG <40), 143 premutation (PM) (CGG 56-170), 197 full mutation (FM) (CGG 200-2000), and 33 CGG size and methylation mosaic samples. RESULTS: In male and female newborn blood spots, MS-QMA differentiated FM from control alleles, with sensitivity, specificity, and positive and negative predictive values between 92% and 100%. In venous blood of FM females between 6 and 35 years of age, MS-QMA correlated most strongly with verbal IQ impairment (P = 0.002). In the larger cohort of males and females, MS-QMA correlated with reference methods Southern blot and MALDI-TOF mass spectrometry (P < 0.05), but was not significantly correlated with age. Unmethylated alleles in high-functioning FM and PM males determined by both reference methods were also unmethylated by MS-QMA. CONCLUSIONS: MS-QMA has an immediate application in FXS diagnostics, with a potential use of its quantitative methylation output for prognosis in both sexes.

Droplet dynamics affecting the shape of patterns formed spontaneously by transforming UV-curable emulsions.

Forming large pitch and depth patterns spontaneously based on a bottom-up approach is a challenging task but with great industrial value. It is possible to spontaneously form an uneven (concave-convex) patterns with submillimeter-to-millimeter-scale pitches and depths by the direct pattern exposure of a UV-curable oil-in-water (O/W) emulsion liquid film. UV irradiation generates a latent pattern of a cured particle aggregation in the liquid film, and an uneven structure is spontaneously formed during the subsequent drying process. This process does not require any printing and embossing plates or development process. In this report, we presented an example of unevenness formation with a maximum pattern depth of approximately 0.4 mm and a maximum pitch width of 5 mm. The patterns formed by this method have raised edges in the exposed areas and fogging in unexposed areas. The pattern shapes become conspicuous under overexposure conditions, but the formation mechanism has not yet been understood in detail and needs to be investigated. In this study, we focused on the exposure process and clarified the mechanism of pattern formation by analyzing the dynamics of emulsion droplets in the medium by an in situ microscopy observation method. As a result, we found that the fogging was mainly caused by light leakage from the exposed area, and the raised pattern edges were caused by droplets transported from the unexposed area to the exposed area. Furthermore, the convection caused by the heat generated from polymerization is a determining factor affecting all these phenomena. By controlling the pattern shape related to convection utilizing direct projection exposure, we showed an example of eliminating raised pattern edges with a height difference of approximately 0.1 mm. By devising and selecting exposure methods, we can expand the range of design applications such as interior decorative patterns.

Fragile X-related element 2 methylation analysis may provide a suitable option for inclusion of fragile X syndrome and/or sex chromosome aneuploidy into newborn screening: a technical validation study.

PURPOSE: We show that a novel fragile X-related epigenetic element 2 FMR1 methylation test can be used along with a test for sex-determining region Y (SRY) to provide the option of combined fragile X syndrome and sex chromosome aneuploidy newborn screening. METHODS: Fragile X-related epigenetic element 2, SRY, and FMR1 CGG repeat analyses were performed on blood and saliva DNA, and in adult and newborn blood spots. The cohort consisted of 159 controls (CGG <40), 187 premutation (CGG 56-170), and 242 full-mutation (CGG ~200-2,000) males and females, 106 sex chromosome aneuploidy individuals, and 151 cytogenetically normal controls. RESULTS: At the 0.435 threshold, fragile X-related epigenetic element 2 analysis in males was robust on both blood DNA and newborn blood spots, with specificity and sensitivity of ~100% for full-mutation genotype. In females, the specificity was 99%, whereas half of full-mutation females were above the 0.435 threshold in both blood DNA and newborn blood spots. Furthermore, at this threshold, the test could not differentiate individuals with Klinefelter syndrome from female controls without using the SRY marker. When combined with SRY analysis, the test was consistent with most results for sex chromosome aneuploidies from karyotyping. CONCLUSION: Setting specific thresholds for fragile X-related epigenetic element 2 analysis and including the SRY marker provides the option to either include or exclude detection of sex chromosome aneuploidies as part of fragile X syndrome newborn screening.

Fragile X mental retardation 1 (FMR1) intron 1 methylation in blood predicts verbal cognitive impairment in female carriers of expanded FMR1 alleles: evidence from a pilot study.

BACKGROUND: Cognitive status in females with mutations in the FMR1 (fragile X mental retardation 1) gene is highly variable. A biomarker would be of value for predicting which individuals were liable to develop cognitive impairment and could benefit from early intervention. A detailed analysis of CpG sites bridging exon 1 and intron 1 of FMR1, known as fragile X-related epigenetic element 2 (FREE2), suggests that a simple blood test could identify these individuals. METHODS: Study participants included 74 control females (<40 CGG repeats), 62 premutation (PM) females (55-200 CGG repeats), and 18 full-mutation (FM) females assessed with Wechsler intelligence quotient (IQ) tests. We used MALDI-TOF mass spectrometry to determine the methylation status of FREE2 CpG sites that best identified low-functioning (IQ <70) FM females (>200 CGG repeats), compared the results with those for Southern blot FMR1 activation ratios, and related these assessments to the level of production of the FMR1 protein product in blood. RESULTS: A methylation analysis of intron 1 CpG sites 10-12 showed the highest diagnostic sensitivity (100%) and specificity (98%) of all the molecular measures tested for detecting females with a standardized verbal IQ of <70 among the study participants. In the group consisting of only FM females, methylation of these sites was significantly correlated with full-scale IQ, verbal IQ, and performance IQ. Several verbal subtest scores showed strong correlation with the methylation of these sites (P = 1.2 × 10(-5)) after adjustment for multiple measures. CONCLUSIONS: The data suggest that hypermethylation of the FMR1 intron 1 sites in blood is predictive of cognitive impairment in FM females, with implications for improved fragile X syndrome diagnostics in young children and screening of the newborn population.

Spontaneous patterning method utilizing transformation of UV-curable emulsion.

A self-organizing structure is important for imparting functions and simplifying the manufacturing process. The development of spontaneous structures with a roll-to-roll process capability is a challenging task. We propose a novel patterning method utilizing the destruction of the emulsion structure. An oil-in-water (O/W) UV-curable emulsion liquid film was partially exposed to UV, resulting in aggregation and immobilization of the emulsion in the medium. Emulsion droplets in the unexposed area are coalesced by removing water. Coalesced emulsion droplets expand and spontaneously permeate into the pores formed in the aggregated structure of UV-cured emulsion particles, causing an uneven structure. An uneven pattern can be formed by direct UV exposure of the liquid film and the subsequent drying process without requiring a development process.

Specificity of expression of the GUS reporter gene (uidA) driven by the tobacco ASA2 promoter in soybean plants and tissue cultures.

Twelve independent lines were transformed by particle bombardment of soybean embryogenic suspension cultures with the tobacco anthranilate synthase (ASA2) promoter driving the uidA (beta-glucuronidase, GUS) reporter gene. ASA2 appears to be expressed in a tissue culture specific manner in tobacco (Song H-S, Brotherton JE, Gonzales RA, Widholm JM. Tissue culture specific expression of a naturally occurring tobacco feedback-insensitive anthranilate synthase. Plant Physiol 1998;117:533-43). The transgenic lines also contained the hygromycin phosphotransferase (hpt) gene and were selected using hygromycin. All the selected cultures or the embryos that were induced from these cultures expressed GUS measured histochemically. However, no histochemical GUS expression could be found in leaves, stems, roots, pods and root nodules of the plants formed from the embryos and their progeny. Pollen from some of the plants and immature and mature seeds and embryogenic cultures initiated from immature cotyledons did show GUS activity. Quantitative 4-methylumbelliferyl-glucuronide (MUG) assays of the GUS activity in various tissues showed that all with observable histochemical GUS activity contained easily measurable activities and leaves and stems that showed no observable histochemical GUS staining did contain very low but measurable MUG activity above that of the untransformed control but orders of magnitude lower than the constitutive 35S-uidA controls used. Low but clearly above background levels of boiling sensitive GUS activity could be observed in the untransformed control immature seeds and embryogenic cultures using the MUG assay. Thus in soybean the ASA2 promoter drives readily observable GUS expression in tissue cultures, pollen and seeds, with only extremely low levels seen in vegetative tissues of the plants. The ASA2 driven expression seen in mature seed was, however, much lower than that seen with the constitutive 35S promoter; less than 2% in seed coats and less than 0.13% in cotyledons and embryo axes. The predominate tissue culture specific expression pattern of the ASA2 promoter may be useful for genetic transformation of crops.

Novel methylation markers of the dysexecutive-psychiatric phenotype in FMR1 premutation women.

OBJECTIVE: To examine the epigenetic basis of psychiatric symptoms and dysexecutive impairments in FMR1 premutation (PM: 55 to 199 CGG repeats) women. METHODS: A total of 35 FMR1 PM women aged between 22 and 55 years and 35 age- and IQ-matched women controls (CGG <45) participated in this study. All participants completed a range of executive function tests and self-reported symptoms of psychiatric disorders. The molecular measures included DNA methylation of the FMR1 CpG island in blood, presented as FMR1 activation ratio (AR), and 9 CpG sites located at the FMR1 exon1/intron 1 boundary, CGG size, and FMR1 mRNA levels. RESULTS: We show that FMR1 intron 1 methylation levels could be used to dichotomize PM women into greater and lower risk categories (p = 0.006 to 0.037; odds ratio = 14-24.8), with only FMR1 intron 1 methylation, and to a lesser extent AR, being significantly correlated with the likelihood of probable dysexecutive or psychiatric symptoms (p < 0.05). Furthermore, the significant relationships between methylation and social anxiety were found to be mediated by executive function performance, but only in PM women. FMR1 exon 1 methylation, CGG size, and FMR1 mRNA could not predict probable dysexecutive/psychiatric disorders in PM women. CONCLUSIONS: This is the first study supporting presence of specific epigenetic etiology associated with increased risk of developing comorbid dysexecutive and social anxiety symptoms in PM women. These findings could have implications for early intervention and risk estimate recommendations aimed at improving the outcomes for PM women and their families.

Expression of a feedback insensitive anthranilate synthase gene from tobacco increases free tryptophan in soybean plants.

Soybean [Glycine max (L.) Merr.] embryogenic cultures were transformed by particle bombardment with the feedback-insensitive tobacco anthranilate synthase (AS) gene ASA2 driven by the CaMV 35S promoter and selected using hph as the selectable marker gene. Only one of eight regenerated lines that set seed and contained ASA2 expressed the gene highly and contained increased free tryptophan (Trp) levels in leaves, seeds and embryogenic cultures. Leaf extracts of the ASA2 expressing line contained about twice as much AS enzyme activity as the untransformed control and this activity was only slightly more feedback-insensitive. Amino acid analysis showed that both leaves and embryogenic tissue cultures of the ASA2 expressing line had four to five-times the normal levels of free Trp and slightly higher free tyrosine and phenylalanine. The seed total Trp content was only slightly increased. Metabolic profiling-analysis by GC-MS detected no other consistent differences. These studies show that the ASA2 gene can be expressed in soybean and that modest changes in Trp synthesis occurs.